Escherichia coli mutants and methods of use thereof

ABSTRACT

The present invention provides methods and compositions for production of gram-negative bacterial mutants that are defective in intestinal colonization capacity and sensitive to infection by bacteriophage P1. Thus the present invention provides immunogenic compositions for the prevention or attenuation of food- and water-borne illnesses associated with ingestion of bacteria such as enterohemorrhagic  Escherichia coli.

This invention was made in part with support under Grant Number R21A1067827 awarded by the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention provides methods and compositions for productionof gram-negative bacterial mutants that are defective in intestinalcolonization capacity and sensitive to infection by bacteriophage P1.Thus the present invention provides immunogenic compositions for theprevention or attenuation of food- and water-borne illnesses associatedwith ingestion of bacteria such as enterohemorrhagic Escherichia coli.

BACKGROUND OF THE INVENTION

Enterohemorrhagic Escherichia coli (EHEC) is an emerging food- andwater-borne pathogen that colonizes the distal ileum and colon andproduces potent cytotoxins (Donnenberg, “Infections due to Escherichiacoli and other enteric gram-negative bacilli,” in ACP Medicine, WebMDProfessional Publishing, Danbury Conn., Chapter 7, pp. 8-1 to 8-18,2005). After ingestion of contaminated food, humans can develop symptomsranging from mild diarrhea to the severe, and at times life-threatening,hemolytic uremic syndrome (HUS). Currently, EHEC is the most commoncause of pediatric renal failure in the United States (Mead et al.,Emerg Infect Dis, 5:607-625, 1999). Several EHEC serotypes causedisease, but the O157 serotype is by far the most common cause ofEHEC-related disease in North America, Europe and Japan (Feng,“Escherichia coli,” in Garcia (ed.) Guide to Foodborne Pathogens, JohnWiley and Sons, Inc., pp. 143-162, 2001). EHEC O157:H7 colonization ofhealthy cattle remains a serious public health threat due to the lownumbers of EHEC O157:H7 (e.g., 10-100) necessary to infect a human andto the bulk processing of slaughtered cattle. Methods for detecting andsubsequently killing EHEC O157:H7 at slaughter, altering the diet ofcattle to reduce the number of intestinal EHEC O157:H7 and immunizinganimals to prevent EHEC O157:H7 colonization are being employed toaddress this problem. Recently, the recombinant production and use ofEHEC O157:H7 proteins including recombinant EspA (InternationalPublication No. WO 97/40063), recombinant TIR (International PublicationNo. WO 99/24576), recombinant EspB and recombinant Initimin (Li et al.,Infec Immun, 68:5090-5095, 2000) have been described. However,production and purification of recombinant proteins in amountssufficient for use as antigens is difficult and expensive.

Thus there is a need in the art for compositions and method for blockingEHEC O157:H7 colonization of cattle and other mammals and, thereby, forreducing shedding of EHEC into the environment. These tools arecontemplated to be useful for reduce the incidence of health problemsassociated with EHEC-contaminated meat and water.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for productionof gram-negative bacterial mutants that are defective in intestinalcolonization capacity and sensitive to infection by bacteriophage P1.Thus the present invention provides immunogenic compositions for theprevention or attenuation of food- and water-borne illnesses associatedwith ingestion of bacteria such as enterohemorrhagic Escherichia coli.

In particular the present invention provides an isolated Escherichiacoli (E. coli) bacterium comprising an inactivating mutation in one ormore galactose-modifying enzymes. In some embodiments, thegalactose-modifying enzymes are selected from the group consisting ofGalE, GalT, GalK and GalU. In preferred embodiments, E. coli is selectedfrom the group consisting of enterohemorrhagic E. coli (EHEC),enteropathogenic. E. coli (EPEC), enterotoxigenic E. coli (ETEC) anduropathogenic E. coli (UPEC). In a subset of these embodiments, the EHECis E. coli O157. In some preferred embodiments, the E. coli O157 isserotype O157:H7. In some particularly preferred embodiments, the E.coli O157 is of a strain selected from the group consisting of TEA007(galE::pTHE001), TEA023 (ΔgalU::aad-7), TEA026 (ΔgalETKM::aad-7) andTEA028 (ΔgalETKM::tetA). In preferred embodiments, the inactivatingmutation is associated with one or more of reduced O-antigen expression,with increased susceptibility to bacteriophage P1, with increasedsensitivity to bactericidal/permeability-increasing protein (BPI) andreduced intestinal colonization. In some embodiments, the BPI comprisesthe amino acid sequence of peptide P2 set forth as SEQ ID NO:9. Thepresent invention also provides E. coli bacterium further comprising aninactivating mutation in one or both of a shiga toxin A subunit and ashiga toxin B subunit. In some embodiments, the shigatoxin A subunit isselected from the group consisting of Stx1A and Stx2A. In some preferredembodiments, the E. coli bacterium further comprises a heterologousantigen. In particularly preferred embodiments, the heterologous antigencomprises a protein encoded by a bacterial, viral or protozoal pathogen,which in some embodiments is a human pathogen. Exemplary heterologousantigens include but are not limited to cholera antigens; HIV-1 antigensand Toxoplasma gondii antigens. In a subset of these embodiments, the E.coli bacterium comprises a live bacterial culture.

In addition the present invention provides compositions comprising an E.coli bacterium comprising an inactivating mutation in one or moregalactose-modifying enzymes, suspended in an adjuvant or an excipient.In preferred embodiments, the E. coli bacterium further comprises aninactivating mutation in one or both of a shiga toxin A subunit and ashiga toxin B subunit, Furthermore the present invention providesmethods for inducing an immune response comprising administering theclaimed compositions to a subject under conditions suitable for inducingan immune response against the E. coli bacterium. In some embodiments,the administering is done orally or intrarectally. In some preferredembodiments, the subject is a human, while in others the subject is aruminant. In particularly preferred embodiments, the ruminant is abovine subject.

Moreover the present invention provides methods for reducing intestinalcolonization of EHEC in a subject comprising administering a compositioncomprising an E. coli bacterium comprising an inactivating mutation inone or more galactose-modifying enzymes, and an inactivating mutation inone or both of a shiga toxin A subunit and a shiga toxin B subunit, tothe subject under conditions suitable for reducing intestinalcolonization. In some preferred embodiments, the subject is a human,while in others the subject is a ruminant. In preferred embodiments, theintestinal colonization comprises one or more of ileum colonization,mid-colon colonization and cecum colonization.

The present invention also provides methods for reducing fecal sheddingof EHEC by a subject comprising administering a composition comprisingan E. coli bacterium comprising an inactivating mutation in one or moregalactose-modifying enzymes, and an inactivating mutation in one or bothof a shiga toxin A subunit and a shiga toxin B subunit, to the subjectunder conditions suitable for reducing fecal shedding. In some preferredembodiments, the subject is a human, while in others the subject is aruminant.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the results of in vivo and in vitro competition assays ofan EHEC galETKM− mutant (TEA026) versus wild type (EDL933) or versus aGal+ revertant of TEA026 (TEA040). In FIG. 1A equal numbers of TEA026and EDL933 and in FIG. 2B equal numbers of TEA026 and TEA040 wereco-inoculated into infant rabbits or into LB. After seven days ofinfection, sections of the ileum, mid-colon and cecum and samples of thestool were processed to determine competitive indeces. The in vitrocompetition assays in LB were carried out for 16 hours. CompetitiveIndex=[(number of mutant bacteriaoutput)/(number of wild type orrevertant bacteriaoutput)]/[(number of mutant bacteriainput)/(number ofwild type or revertant bacteriainput)]. Each of the competitive indecesshown is statistically different from 1 (p<0.00001 using the student'st-test).

FIG. 2 illustrates an exemplary scheme for P1-facilitated geneticengineering of EHEC. FIG. 2A shows the construction of the desiredmutation in the ΔgalETKM::tetA or ΔgalETKM::aad-7 background (e.g. usingthe lambda red recombination system as shown in the figure) so that themutation can be moved by P1 transduction. FIG. 2B shows the movement ofthe desired mutation into the galE::pTHE001 background using P1transduction selecting for abR to generate an isogenic back-crossedmutant. FIG. 2C shows a second P1 transduction into the back crossedstrain followed by selecting for growth on galactose minimal plates.Abbreviations: x=gene of interest, abR=antibiotic resistance gene,tetR=tetracycline resistance gene.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “purified” and “isolated” refer to molecules(polynucleotides or polypeptides) or organisms that are removed orseparated from their natural environment. “Substantially purified”molecules or organisms are at least 50% free, preferably at least 75%free, more preferably at least 90% and most preferably at least 95% freefrom other components with which they are naturally associated.

The term “wild-type” refers to a gene, gene product or organism that hasthe characteristics of that gene, gene product or organism when isolatedfrom a naturally occurring source. A wild type gene or organism is thatwhich is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the gene ororganism.

In contrast, the terms “modified,” “mutant,” and “variant” refer to agene, gene product or organism that displays modifications in sequenceand or functional properties (i.e., altered characteristics) whencompared to the wild-type gene, gene product or organism. It is notedthat naturally occurring mutants can be isolated; these are identifiedby the fact that they have altered characteristics when compared to thewild-type gene, gene product or organism.

The term “antibody” refers to polyclonal and monoclonal antibodies.Polyclonal antibodies which are formed in the animal as the result of animmunological reaction against a protein of interest or a fragmentthereof, can then be readily isolated from the blood using well-knownmethods and purified by column chromatography, for example. Monoclonalantibodies can also be prepared using known methods (See, e.g., Winterand Milstein, Nature, 349, 293-299, 1991). As used herein, the term“antibody” encompasses recombinantly prepared, and modified antibodiesand antigen-binding fragments thereof, such as chimeric antibodies,humanized antibodies, multifunctional antibodies, bispecific oroligo-specific antibodies, single-stranded antibodies and F(ab) orF(ab)₂ fragments. The term “reactive” in used in reference to anantibody indicates that the antibody is capable of binding an antigen ofinterest. For example, an EHEC-reactive antibody is an antibody thatbinds to EHEC.

As used herein, the term “immune response” refers to the reactivity of asubject's immune system in response to an antigen. In mammals, this mayinvolve antibody production, induction of cell-mediated immunity, and/orcomplement activation. In preferred embodiments, the term immuneresponse encompasses but is not limited to one or more of a “lymphocyteproliferative response,” a “cytokine response,” and an “antibodyresponse.”

In particularly preferred embodiments, the immune response is largelyreactive with EHEC cells. For instance, when used in reference toadministration of EHEC gal mutants to a subject, the term refers to theimmune response produced in the subject that reacts with the EHEC cells.Immune responses reactive with EHEC cells are measured in vitro usingvarious methods disclosed herein.

The term “reactive with an antigen of interest” when made in referenceto an immune response refers to an increased level of the immuneresponse to the antigen of interest (e.g., EHEC) as compared to thelevel of the immune response to a control (e.g., irrelevant antigen).

The term “lymphocyte proliferative response” refers to EHEC-inducedincrease in lymphocyte numbers. Alternatively, or in addition, the term“proliferation” refers to the physiological and morphologicalprogression of changes that cells undergo when dividing, for instanceincluding DNA replication as measured by tritiated thymidineincorporation.

The term “cytokine response” refers to EHEC-induced cytokine secretionby lymphocytes as measured for instance by assaying culture supernatantsfor cytokine content (e.g., IL-2, IFNγ, TNFα, IL-4, etc) by ELISA.

The term “antibody response” refers to the production of antibodies(e.g., IgM, IgA, IgG) that bind to an antigen of interest (e.g., EHECcells), this response is measured for instance by assaying sera by EHECELISA.

The term “adjuvant” as used herein refers to any compound that wheninjected together with an antigen, non-specifically enhances the immuneresponse to that antigen. Exemplary adjuvants include but are notlimited to incomplete Freunds adjuvant (IFA), aluminum-based adjuvants(e.g., AIOH, AIPO4, etc), and Montanide ISA 720.

The terms “excipient,” “carrier” and “vehicle” as used herein refer tousually inactive accessory substances into which a pharmaceuticalsubstance (e.g., EHEC cells) is suspended. Exemplary carriers includeliquid carriers (such as water, saline, culture medium, aqueousdextrose, and glycols) and solid carriers (such as carbohydratesexemplified by starch, glucose, lactose, sucrose, and dextrans,anti-oxidants exemplified by ascorbic acid and glutathione, andhydrolyzed proteins).

The terms “mammals” and “mammalian” refer to animals of the classmammalian that nourish their young by fluid secreted from mammary glandsof the mother, including human beings. The class “mammalian” includesplacental animals, marsupial animals, and monotrematal animals.

The term “ruminant” as used herein refers to animals of the suborderRuminantia or any other animal that chews a cud. In preferredembodiments, the term ruminant encompasses cattle, goats, sheep, bison,buffalo, deer, and antelope.

The term “bovine subject” as used herein refers to animals belonging tothe genus Bos. In preferred embodiments, the term bovine encompassescattle.

The term “control” refers to subjects or samples that provide a basisfor comparison for experimental subjects or samples. For instance, theuse of control subjects or samples permits determinations to be maderegarding the efficacy of experimental procedures. In some embodiments,the term “control subject” refers to animals or cells receiving a mocktreatment (e.g., adjuvant alone).

As used herein the terms “GalE” and “UDP-galactose-4-epimerase” refer toan enzyme (EC 5.1.3.2) that catalyzes the interconversion of UDP-glucoseto UDP-galactose and the interconversion of UDP-N-acetylglucosamine toUDP-N-acetylgalactosamine. In an exemplary embodiment the term GalUrefers to an E. coli O157:H7 enzyme having an amino acid sequence as setforth in GENBANK Accession No. NP_(—)286480, herein incorporated byreference, encoded by a nucleic acid sequence complementary to residues876559 to 877575 of GENBANK Accession No. NC_(—)002655.2 hereinincorporated by reference.

As used herein the terms “GalT” and “galactose-1-phophateuridylyltransferase” refer to an enzyme (EC 2.7.7.12) that catalyzes theinterconversion of galactose-1-phosphate and glucose-1-phosphate viatransfer of uridine monophosphate. In an exemplary embodiment the termGalT refers to an E. coli O157:H7 enzyme having an amino acid sequenceas set forth in GENBANK Accession No. NP_(—)286479, herein incorporatedby reference, encoded by a nucleic acid sequence complementary toresidues 875503 to 876549 of GENBANK Accession No. NC_(—)002655.2 hereinincorporated by reference.

As used herein the terms “GalK” and “galactokinase” refer to an enzyme(EC 2.7.1.6) that catalyzes the phosphorylation of galactose togalactose-1-phosphate as the first step in galactose metabolism. In anexemplary embodiment the term GalK refers to an E. coli O157:H7 enzymehaving an amino acid sequence as set forth in GENBANK Accession No.NP_(—)286478, herein incorporated by reference, encoded by a nucleicacid sequence complementary to residues 874351 to 875499 of GENBANKAccession No. NC_(—)002655.2 herein incorporated by reference.

As used herein the terms “GalU,” “glucose-1-phosphateuridylyltransferase” and UGP refer to an enzyme (EC 2.7.7.9) thatcatalyzes the transfer of a glucose moiety from glucose-1-phosphate toMgUTP, forming UDP-glucose and MgPPi. In an exemplary embodiment theterm GalU refers to an E. coli O157:H7 enzyme having an amino acidsequence as set forth in GENBANK Accession No. NP_(—)287481, hereinincorporated by reference, encoded by a nucleic acid sequence as setforth in residues 1828438 to 1829346 of GENBANK Accession No.NC_(—)002655.2 herein incorporated by reference.

As used herein the terms “shiga toxin 1 subunit A,” “Stx1A” and“shiga-like toxin 1 subunit A” refers to the A subunit of an A-B typetoxin that inhibits protein synthesis in eukaryotic cells and is thoughtto be required for the severe clinical manifestations of EHEC infection,such as hemorrhagic colitis and HUS (Karmali, Clin Microbiol Rev,2:15-38, 1989). In an exemplary embodiment the term Stx1A refers to anE. coli O157:H7 protein having an amino acid sequence as set forth inGENBANK Accession No. NP_(—)288673, herein incorporated by reference,encoded by a nucleic acid sequence complementary to residues 2996033 to2996980 of GENBANK Accession No. NC_(—)002655.2 herein incorporated byreference.

As used herein the terms “shiga toxin 2 subunit A,” “Stx2A” and“shiga-like toxin 2 subunit A” refers to the A subunit of an A-B typetoxin that inhibits protein synthesis in eukaryotic cells and is thoughtto be required for the severe clinical manifestations of EHEC infection,such as hemorrhagic colitis and HUS (Karmali, Clin Microbiol Rev,2:15-38, 1989). In an exemplary embodiment the term Stx2A refers to anE. coli O157:H7 protein having an amino acid sequence as set forth inGENBANK Accession No. NP 286976, herein incorporated by reference,encoded by a nucleic acid sequence as set forth in residues 1352290 to1353249 of GENBANK Accession No. NC_(—)002655.2 herein incorporated byreference.

BRIEF DESCRIPTION OF THE INVENTION

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7 isan emerging cause of food-borne illness. Prior to development of thepresent invention, E. coli O157 could not be genetically manipulatedusing the generalized transducing phage P1, presumably because itsO-antigen obscures the P1 receptor, the lipopolysaccharide (LPS) coresubunit. The GalE, GalT, GalK and GalU proteins are necessary formodifying galactose before it can be assembled into the repeatingsubunit of the O-antigen. As disclosed herein E. coli O157:H7 galmutants were constructed having little or no O-antigen. These strainswere able to adsorb P1. P1 lysates grown on the gal strains could beused to move chromosomal markers between EHEC strains, therebyfacilitating genetic manipulation of E. coli O157:H7. The gal mutantscould easily be reverted to a wild type Gal+ strain using P1transduction. The O157:H7 galETKM::aad-7 deletion strain was 500-foldless able to colonize the infant rabbit intestine compared to theisogenic Gal+ parent, although it displayed essentially no growth defectin vitro. Furthermore, a Gal+ revertant of this mutant out-competed thegalETKM deletion strain to a similar extent as wild type indicating thatthe O157 O-antigen is an important intestinal colonization factor.Compared to the wild type, EHEC gal mutants were 100-fold more sensitiveto a peptide derived from bactericidal/permeability-increasing protein(BPI), a bactericidal protein found on the surface of intestinalepithelial cells. Thus, the EHEC gal mutants are sensitive tohost-derived anti-microbial polypeptides.

DETAILED DESCRIPTION OF THE INVENTION I. O-Antigen Mutants of EHEC

Lipopolysaccharide (LPS), found in the outer membrane of gram-negativebacteria, is composed of lipid A, core oligosaccharide and repeatingO-antigen subunits. The O-antigen is covalently linked to the outerregion of the core oligosaccharide, and it appears to act as a barrierthat can protect enteric pathogens against toxic agents encountered inhost gastrointestinal (GI) tracts (Peschel, Trends Microbiol,10:179-186, 2002). For example, in Vibrio cholerae, galU and galEmutants lacking O-antigen are defective in intestinal colonizationalthough they have no growth defect in rich medium. These mutants weremore sensitive than O-antigen-producing strains to killing by complementand cationic anti-microbial peptides, suggesting that their defect incolonization is attributable to their sensitivity to bactericidalsubstances elaborated by the host GI tract (Nesper et al., Infect Immun,69:435-445, 2001).

Like many enteric pathogens, E. coli O157 produces LPS that contains anextensive O-antigen. The O157 O-antigen subunit consists ofN-acetyl-D-perosamine, L-fucose, D-glucose, and N-acetyl-D-galactose(Perry et al., Biochem Cell Biol, 64:21-28, 1986). Production ofN-acetyl-D-galactose requires its precursor, galactose, be modified bythe enzymes GalE, GalT, GalK and GalU. Salmonella enterica serovarTyphimurium (S. Typhimurium) and E. coli gal mutants no longer makeO-antigen (Genevaux et al., Arch Micrbiol, 172:1-8, 1999; Ornellas etal., Virol, 60:491-502, 1974; and Raetz, “Bacterial lipopolysaccharides:a remarkable family of bioactive macroamphilies,” in Neidhardt (ed.),Escherichia coli and Salmonella, vol. 1, ASM Press, pp 1035-1063, 1996).The inner region of the LPS core oligosaccharide, which is conserved inmany enterics, serves as the receptor for bacteriophage P1. Phage P1 hasbeen a workhorse for genetic manipulation of E. coli K-12 for manydecades. P1-mediated generalized transduction enables movement ofmutations for generation of isogenic bacterial strains, which is oftenrequired for proving the linkage between particular genotypes andphenotypes. In S. Typhimurium, which has a LPS core oligosaccharidesimilar to that of E. coli K-12, the long O-antigen obscures the coreoligosaccharide and prevents P1 from adsorbing to the bacteria.O-antigen mutants (Δgal, ΔgalE, and ΔgalU) of S. Typhimurium have beenshown to be P1-sensitive (Ornellas et al., Virol, 60:491-502, 1974).

II. P1-Mediated Generalized Transduction of EHEC O-Antigen Mutants

Generalized transduction of EHEC by P1 has not been previouslydescribed. As disclosed herein, O157:H7 gal mutants were produced, whichare P1 sensitive and permit P1-mediated movement of genetic markersbetween EHEC strains. In contrast, wild type EHEC O157:H7 are resistantto P1. Additionally, the methods described herein allow for a simplereversion to convert P1 sensitive strains back to wild type P1 resistantstrains.

The methods described herein are adaptable for use with other EHECserotypes, as well as for other pathogenic E. coli such asenteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), anduropathogenic E. coli (UPEC). An exemplary EPEC strain is UT189 (Mulveyet al., Infect Immun, 69:4572-4579, 2001). An exemplary ETEC strain isserotype O78:H11, LT+, ST+, strain H10407 (Evans et al., J Infect Dis,136(S):S118-S123, 1977. An exemplary EPEC strain is 0127:H6 wild typestrain E2348/69 (Jerre et al., Proc Natl Acad Sci USA, 87:7839-7843,1990).

FIG. 2 outlines a general scheme for using P1 transduction in thegenetic manipulation of EHEC. This figure describes methods forroutinely generating isogenic EHEC strains. This ability to movechromosomal markers enables generation of isogenic strains containingsingle or multiple mutations. Given the relative ease of the P1transduction technique outlined here, it is now possible to back crossmutations generated with the lambda red system into geneticallyidentical backgrounds.

III. Colonization Defect of EHEC O-Antigen Mutants

Interestingly, P1-sensitive, galETKM O157:H7 mutant was extremelyattenuated in its ability to colonize the infant rabbit intestine. Thusduring development of the present invention, the O157 O-antigen wasfound to be an important EHEC colonization factor. Tissue cultureexperiments indicate that the O157 O-antigen is not likely to beinvolved in EHEC adherence to intestinal epithelial cells and may reduceEHEC type 3 secretion of effectors that promote adherence (Bilge et al.,Infect Immun, 64:4795-4801, 1996; Cockerill et al., Infect Immun,64:3196-3200, 1996; and Paton et al., Microbial Pathogenesis, 24:57-63,1998). The sensitivity of the O157 gal mutants to BPI indicates that atleast part of the colonization defect of the galETKM mutant is due toits reduced resistance to host anti-microbial factors. Nonetheless,knowledge of mechanism is not required in order to make and use thepresent invention.

Although O157 strains are most commonly associated with EHEC-relatedillness in North America, Europe and Japan, other EHEC serotypes (O26,O91, O103, and O111) can also cause significant human disease (Paton etal., Clin Microbiol Rev, 11:450-479, 1998). It is contemplated thatO-antigens from different EHEC serotypes vary in resistance to hostkilling and therefore vary in intestinal colonization capacity as hasbeen seen in K1 E. coli (Pluschke et al., Infect Immun, 43:684-692,1998). The P1 transduction method outlined herein should facilitate theconstruction of isogenic strains differing only in their O-antigens,thus enabling a more extensive exploration of the role of particularO-antigens in EHEC virulence.

O-antigens are contemplated to act as important barriers againstextracellular assaults mounted by the host immune system. O-antigenmutants of enterics like V. cholerae, Klebsiella serotype O1:K20, andShigella flexneri are more susceptible to anti-microbial peptides andcomplement killing (McCallum et al., Infect Immun, 57:3816-3822, 1989;Nesper et al., Infect Immun, 69:435-445, 2001; and West et al., Science,307:1313-1317, 2005). Additionally, S. Typhi, Brucella melitensis and V.cholerae O-antigen mutants have been shown to be impaired in intestinalcolonization (Chiang et al., Infect Immun, 67:976-980, 1999; Gilman etal., J Infect Dis, 136:717-723, 1997; Nesper et al., Infect Immun,69:435-445, 2001; and Rajashekara et al., Infect Immun, 74:2925-2936,2006). In fact, the only oral live-attenuated vaccine against Salmonellaenterica serovar Typhi used in the United States is a galE mutant. Giventhe pronounced attenuation of the O157:H7 ΔgalETKM::aad-7 mutant, EHECgal mutants are contemplated to be clinically and agriculturally usefulvaccines.

IV. Utilities

In some embodiments, the present invention further provides E. colibacterium comprising an inactivating mutation in one or moregalactose-modifying enzymes, as well as an inactivating mutation in oneor both of a shiga toxin A subunit and a shiga toxin B subunit. Thus thepresent invention provides live attenuated bacteria having defects inintestinal colonization and which are relatively nonpathogenic for useas vaccines and delivery vehicles for genes and gene products and tomethods for their preparation. In some preferred embodiments, thepresent invention provides E. coli O157:H7 gal stx mutants and methodsfor their production and use.

In particular the present invention provides methods for producing anattenuated bacterium comprising (a) introducing one or more inactivatingmutations in a galactose-modifying enzyme; and (b) introducing one ormore inactivating mutations in a shiga toxin, wherein steps (a) and (b)may be performed in any order. Furthermore the present inventionprovides methods for inducing an immune response comprisingadministering immunogenic compositions or vaccines comprising live orkilled E. coli bacterium comprising an inactivating mutation in one ormore galactose-modifying enzymes, as well as an inactivating mutation inone or both of a shiga toxin A subunit and a shiga toxin B subunit to asubject under conditions suitable for inducing an immune responsereactive with the E. coli bacterium.

Although live attenuated vaccines based on Salmonella mutants have shownpromise in stimulating humoral, cell-mediated and mucosal immuneresponses to heterologous antigens (Calhoun et al., J Micrbiol ImmunolInfect, 39:92-97, 2006), Salmonella species do not encode an intact typeII secretion system. Many pathogens secrete important virulence factorsvia a type II secretion system (Sandkvist, Infect Immun, 69:3523-3535,2001; and Dorsey et al., Cell Microbiol, 8:1516-1527, 2006), includingcholera toxin from Vibrio cholerae, the heat-labile toxin fromenterotoxigenic E. coli and Exotoxin A of Pseudomonas aeruginosa. SinceEHEC synthesizes a functional type II secretion system, a live oralvaccine derived from EHEC may be made to secrete harmless components ofthese toxins. Such a vaccine is contemplated to induce an immuneresponse that neutralizes the damaging effects of intact toxins.

The present invention also provides methods for producing an attenuatedcarrier bacterium for delivery of a desired gene product to a host,comprising (a) introducing one or more inactivating mutations in agalactose-modifying enzyme; (b) introducing one or more inactivatingmutations in a shiga toxin; and (c) introducing a recombinant geneencoding the desired gene product to a bacterium, wherein steps (a),(b), or (c) may be performed in any order.

Furthermore the present invention provides methods for inducing animmune response comprising administering immunogenic compositions orvaccines comprising live or killed E. coli bacterium comprising aninactivating mutation in one or more galactose-modifying enzymes, aswell as an inactivating mutation in one or both of a shiga toxin Asubunit and a shiga toxin B subunit, and a gene encoding a heterologousantigen, to a subject under conditions suitable for inducing an immuneresponse reactive with the heterologous antigen.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μM (micrometers); nm (nanometers);(degrees Centigrade); U (units), mU (milliunits); min. (minutes); sec.(seconds); % (percent); kb (kilobase); by (base pair); PCR (polymerasechain reaction); BPI (bactericidal/permeability-increasing protein);EHEC (enterohemorrhagic); LPS (lipopolysaccharide).

Example 1 Production of Enterohemorrhagic E. coli O157:H7 gal Mutants

This example describes the construction of O-antigen deficient E. coliO157:H7 mutants. As described herein, the EHEC mutants produced duringdevelopment of the present invention are able to adsorb the generalizedtransducing phage P1 and do not display growth defects in vitro.However, the EHEC mutants poorly colonize the intestine in vivo and aresensitive to bactericidal/permeability-increasing protein (BPD.

Bacterial Strains and Growth. A list of strains used in duringdevelopment of the exemplary EHEC mutants are shown in Table 1. Unlessotherwise noted, strains were grown in LB broth or on LB agar plates.For antibiotic selection, agar plates were supplemented with ampicillin(80 μg/ml), spectinomycin (100 μg/ml) or tetracycline (6 μg/ml).MacConkey plates with 1% galactose or M63 (22 mM KH₂PO₄, 40 mM K₂HPO₄,15 mM (NH₄)₂SO₄, 0.5 mg/liter FeSO₄) agar plates supplemented with 0.2%galactose and 0.1% casamino acids were used to test whether a straincould metabolize galactose.

To generate the deletion-insertion mutations in the gal genes, aone-step gene inactivation method adapted from Datsenko and Wanner (ProcNatl Acad Sci USA, 97:6640-6645, 2000) was used. In this method, atemperature-sensitive plasmid (pKD46) encoding lambda red recombinasewas transformed into EDL933. To make the ΔgalU::aad-7 mutant TEA023 andthe ΔgalETKM::aad-7 mutant TEA026, the spectinomycin resistance gene(aad7) was amplified from the pVi36 plasmid (provided by V. Burrus,University of Sherbrooke) template using primers TE139 (5′-ATGGCTGCCATTAATACGAA AGTCAAAAAA GCC set forth as SEQ ID NO:1) and TE140(5′-TTACTTCTTA ATGCCCATCT CTTCTTCAAG CCA set forth as SEQ ID NO:2) orTE137 (5′-ATGCTATGGT TATTTCATAC CATAAGCCTA ATGGAGCCCG GCGGATTTGT CCTACTCset forth as SEQ ID NO:3) and TE138 (5′-TTACTCAGCA ATAAACTGAT ATTCCGTCAGGCTCTAAGCA CTTGTCTCCT GTTTA set forth as SEQ ID NO:4) respectively. Forthe ΔgalETKM::tetA mutant TEA028, the tetracycline resistance gene(tetA) was amplified from the pAH162 plasmid (Haldimann et al., JBacteriol, 183:6384-6393, 2001) template using TE141 (5′-ATGCTATGGTTATTTCATAC CATAAGCCTA ATGGAGGATG CCTGGCAGTT CCCTACTC set forth as SEQ IDNO:5) and TE142 (5′-TTACTCAGCA ATAAACTGAT ATTCCGTCAG GCTTTAGGTGGCGGTACTTG GGTCGA set forth as SEQ ID NO:6). After electroporation ofthe PCR products, cells were incubated in SOB 0.2% L-arabinose for 2hours then plated on selective media at 37° C. For the ΔgalU::aad-7mutation, the spectinomycin resistance gene replaced all of the galUgene except for the first 33 by and the last 30 by of the galU openreading frame. For the ΔgalETKM::aad-7 and ΔgalETKM::tetA mutations, theantibiotic resistance gene replaced all of the galETKM operon except forthe first 36 by of the galE gene and the last 30 by of the galM gene.The pTHE001 plasmid was constructed to generate an insertion mutation ingalE. First, a 460 by internal fragment of the galE gene was amplifiedby PCR using primers TE013 (5′-GCAAGGATCC GACGTTTGTT GAAGGCGATA setforth as SEQ ID NO:7) and TE014 (5′-GGCATAAGGG AATTCGGAAT GCCTTGCGGA setforth as SEQ ID NO:8). This PCR product was digested with BamHI and thencloned into the BglII site of the conditional plasmid pGP704 (Miller etal., J Bacteriol, 170:2575-2583, 1988). The resulting pTHE001 plasmidwas mobilized using the RP4+ helper strain WM3064 (provided by W.Metcalf, University of Illinois, Urbana-Champaign) into EDL933. Togenerate the Gal+ revertant TEA040, the galE::pTHE001 mutation was movedfrom TEA007 into the galETKM::aad-7 strain (TEA026) by P1 transduction,selecting for ampicillin resistance. The resulting strain was then usedas a recipient for P1 transduction of the galETKM+ allele from TEA023.Gal+ transductants were selected on M63 agar plates supplemented with0.2% galactose and 0.1% casamino acids.

TABLE 1 List of Strains Strains Genotype/Description Source* EDL933O157:H7 Perna et al. TEA007 EDL933 galE::pTHE001 this study TEA023 EDL933 ΔgalU::aad-7 this study TEA026 EDL 933 ΔgalETKM::aad-7 this studyTEA028 EDL 933 ΔgalETKM::tetA this study TEA040 EDL933; Gal+ revertantof TEA026 this study CAG5051 HfrH nadA57::Tn10 thi-1 relA1 spoT supQ80Singer et al. MC4100 araD139 Δ(arg-lac)U169 rpsL150 relA1 flbB5301fruA25 deoC1 laboratory ptsF25 stock EDL933/ (oriR101 bla Para-λ gam betexo) this study pKD46 BW19851/ RP-4-2-Tc::Mu-1 kan::Tn7 integrantcreB150 hsdR17 endA zbf-5 Miller et al. pGP704 uidA(ΔMlu1)::pir(wt)recA1 thi/pGP704 (oriR6K mobRP4 Ap^(R)) WM3064/ thrB1004 pro thi rpsLhsdS lacZΔM15 RP4-1360 Δ(araBAD)567 this study pTHE001 ΔdapA1341::[ermpir(wt)]/pTHE001 (oriR6K mobRP4 Ap^(R) ‘galEEDL933’) DH5αλpir+/ endA1hadR17 thi-1 recA1 Δ(lacIZYA-argF)U169 deoR (p80 Burrus pVi36 lacZΔM15)gyrA relA1/pVi36 (oriR6K aad-7) BW23473/ Δ(lacIZYA-argF)U169 rph-1rpoS396(Am) robA1 creC510 hsdR514 Haldimann pAH162 DendA9 5uidA(ΔMlu1)::pir(wt) recA1/pAH162 (tetA) et al. *Sources include Pernaet al., Nature, 409: 529-533, 2001; Singer et al., Microbiol Mol BiolRev, 53: 1-24, 1989; Miller et al., J Bacteriol, 170: 2575-2583, 1988;and Haldimann et al., J Bacteriol, 183: 6384-6393, 2001.

P1 Adsorption and Sensitivity Assays. P1 adsorption assays wereperformed using a P1 lysate grown on the E. coli K-12 strain MC4100.Approximately 100 μl of overnight culture was pelleted by centrifugationand resuspended in 100 μl of LB broth. 100 μl of the P1 lysate was thenadded to these cells. After 15 minutes of incubation at 37° C., cellswere pelleted by centrifugation at 6,000 rpm in an Eppendorf centrifugeat 4° C. for 2 minutes. The P1 titers of the supernatants were thendetermined by plaquing using MC4100 as an indicator strain. To plaqueP1, phage lysates were spotted on top agar (LB containing 2 mM MgSO₄ and10 mM CaCl₂ and 0.7% agar) lawns of MC4100.

To test for sensitivity of strains to P1 lysis each strain wascross-streaked against P1. A single line of P1 (100 μl; ˜10⁹ pfu/ml) wasallowed to dry on a LB agar plate. For each bacterial strain, a singlestreak was then drawn across the perpendicular line of phage P1. Strainsresistant to P1 grow on both sides of the line of P1, while susceptiblestrains partially lyse following an encounter with P1.

P1 Lysate Production. P1 lysates of various EHEC strains were generatedby growing 1:100 dilution overnight cultures of each strain in 2.5 ml ofLB containing 2 mM MgSO₄ and 10 mM CaCl₂ and incubating for 1 hour at37° C. with agitation. Then, 100 μl of a P1 lysate grown on MC4100 wasadded to each culture. After 2-3 hours of incubation at 37° C. withagitation, lysis of the cultures was observed. Tubes were transferred toice and any remaining intact bacteria were lysed with 0.5 ml chloroform.Lysates were then centrifuged at 13,000 rpm for 1 minute, diluted inphosphate-buffered saline (PBS) and spotted on top agar lawns of MC4100for titering. Lysates were stored at 4° C. in the dark with 0.5 mlchloroform.

P1 Transduction. Overnight cultures (0.5 ml) of recipient bacteria grownin LB were pelleted and resuspended in 100 μl MC (5 mM MgSO₄ 50 mMCaCl₂). About 50 μl of P1 lysate was added to the cells, which were thenincubated at 37° C. for 15-30 minutes. LB with 10 mM sodium citrate(0.5-1 ml) was added to each tube and incubated for 1 hour at roomtemperature. Each tube was centrifuged at 6,000 rpm for 2 minutes,resuspended in 100 μl 1M sodium citrate and plated on selective media.

In Vitro Competition Assays. A 1:1 mixture of mutant (TEA026) and wildtype (EDL933 or TEA040) initially containing (5×10⁷) bacteria/ml wasincubated in LB broth at 37° C. with agitation overnight. Each assay wasthen diluted and plated on LB agar. After overnight growth, bacteriawere replica plated on selective media to determine the number of mutantand wild type bacteria. Each assay was performed at least three times.

Competition Assays in Infant Rabbits. The infant rabbit model was usedto test the colonization ability of EHEC strains (Ritchie et al., InfectImmun, 71:7129-7139, 2003). Three-day old New Zealand white rabbits wereorally inoculated with a 1:1 mixture of TEA026 and wild type (EDL933 orTEA040) containing 2.5×10⁸ total bacteria, which were washed one timeand resuspended in PBS. Seven days after inoculation, rabbits weresacrificed and their gastrointestinal tracts removed. Portions of theileum, mid-colon and cecum were then homogenized, diluted and plated onsorbitol-MacConkey agar. After overnight growth, bacteria were replicaplated on LB agar containing spectinomycin (100 μg/ml) to determine thenumber of TEA026 bacteria.

BPI Sensitivity Assays. The BPI-derived peptide P2(SKISGKWKAQKRFLKMSGNFGC, set forth as SEQ ID NO:9), which retains theanti-bacterial activity of whole protein (Gray et al., Infect Immun,62:2732-2739, 1994; and Little et al., J Biol Chem, 269:1865-1872,1994), was used to assess EHEC sensitivity. For these assays, bacteriagrown overnight in LB were washed once in PBS and resuspended in PBS pH6.2. Then 5×10⁷ bacteria with or without 30 μg P2 (Tufts University CoreFacility) were incubated in 0.5 ml PBS pH 6.2 for 45 minutes at 37° C.After incubation, assays were placed on ice, diluted and plated on LB.Each assay was performed in triplicate and repeated in three independentexperiments.

O157:H7 gal mutants can adsorb P1 and are sensitive to P1 lysis. In manyenteric bacteria, extensive LPS O-antigens obscure the LPS coreoligosaccharide and thereby prevent adsorption of and lysis by phage P1.Synthesis of the O157:H7 O-antigen requires modification of galactose bythe gaff, galT, galK and galU gene products. As described herein phageinfection of and adsorption by the sequenced O157:H7 isolate EDL933 wascompared to that of four gal derivatives: the deletion mutantsΔgalETKM::aad-7 (TEA026), ΔgalETKM::tetA (TEA028) and ΔgalU::aad7(TEA023) and the insertion mutant ΔgalE::pTHE001 (TEA007).

To assess whether gal loci influence infection of EHEC by phage P1,cross streak experiments were performed. A streak of each bacterialstrain was drawn across a perpendicular line of P1, and the consequencesof encountering phage were assessed. There was no change in the growthof wild type EDL933 in response to P1, indicating that this strain isresistant to phage infection. In contrast, all four gal mutants werelysed by phage P1.

To explore why the gal mutants are more susceptible to P1 infection,adsorption of P1 by the mutants was tested. Phage adsorption to the hostbacterium is an essential first step in phage infection. In theseassays, each gal mutant was incubated with P1 and then centrifuged topellet any phage adsorbed to the bacteria. Supernatants were thenassayed to determine the number of unadsorbed P1. As shown in Table 2,the wild type strain adsorbed ˜40% of the phage, suggesting that theremay be some nonspecific interactions between EHEC and P1, since the wildtype strain is resistant to P1 infection and therefore presumed to haveinaccessible core oligosaccharide. However, all four gal mutantsadsorbed ˜95% of the P1. Access to P1's receptor is contemplated to beless impeded in the mutant strains than it is in the wild type strain.Nonetheless, knowledge of the mechanism is not required in order to makeand use the present invention.

TABLE 2 P1 Adsorption and P2 Sensitivity of EHEC strains % Survivalafter P2 Strain % Adsorbed P1¹ challenge² EDL933 (wild type) 27.4 38TEA007 (galE::pTHE001) 93.5 0.054 TEA023 (ΔgalU::aad-7) 96.6 0.29 TEA026(ΔgalETKM::aad-7) 95.2 0.014 TEA028 (ΔgalETKM::tetA) 98.4 0.35 TEA040(Gal+ revertant) 15.6 32 ¹% Adsorbed P1 = the number of P1 plaquesobtained after incubation with bacteria/the number of P1 plaquesobtained after incubation without bacteria × 100%. Each experiment wasperformed in triplicate and the means are shown. The p-value for each ofthe gal mutants versus wild type was less than 0.006. The p-value forTEA040 versus wild type was 0.65. ²% Survival after P2 challenge = thenumber of bacteria after incubation with P2/the number of bacteria afterincubation without P2. Each experiment was performed in triplicate andthe means are shown. The p-value for each of the gal mutants versus thewild type was less than 0.03. The p-value for TEA040 versus the wildtype was 0.10.

Antibiotic resistance markers can be transduced between EHEC O157:H7 galmutants by P1. The EHEC gal mutants were tested to determine whetherthey could serve as recipients or donors in P1 transduction. Asexpected, a nadA::Tn10 marker from an E. coli K-12 strain (CAG5051)could not be transduced into the wild type EDL933 strain by P1. Incontrast, this marker could be transduced into the ΔgalU::aad-7(TEA023), ΔgalETKM::aad-7 (TEA026), and the galE::pTHE001 (TEA007)strains.

Next to determine whether chromosomal markers could be transduced fromthe EHEC gal mutants, each of the mutants was used to generate a P1lysate. The ΔgalU::aad-7, ΔgalETKM::aad-7 and galE::pTHE001 mutationscould readily be transferred into the O157:H7 ΔgalETKM::tetA strain orinto the E. coli K-12 strain MC4100. For each of these EHEC P1transduction experiments, between 10-100 transductants were obtained.Thus as demonstrated herein, O157:H7 gal mutants are capable of beingtransduced by P1 and can be used to generate P1 lysates. However, thefrequencies of transduction between the O157:H7 gal strains were˜100-fold lower than the frequency of P1 transduction between K-12strains. The lower O157:H7 transduction efficiency is contemplated to bedue to the abundance of prophage genes in O157:H7 that may affectreplication or production of P1. Nonetheless, knowledge of the mechanismis not required in order to make and use the present invention.

Reversion of the gal mutant using P1 transduction. Gal-strains werereverted back to Gal+ to study the Gal+ EHEC strains that have beenengineered using P1. The ΔgalETKM::aad-7 mutant (TEA026) was notreverted by transducing the galETKM+ allele from TEA023, indicating thatthe frequency of transduction was too low to obtain a revertant.Therefore, a two-step reversion method was employed. First thegalE::pTHE001 mutation from TEA007 was moved into the galETKM strain(TEA026). This strain was then used as a recipient for P1 transductionof the galETKM+ allele from a donor lysate grown on the ΔgalU strain(TEA023). Gal+ transductants were selected on agar plates made with M63minimal media containing 0.2% galactose and 0.1% casamino acids, andverified as Gal+ on MacConkey 1% galactose agar. The frequency of thistransduction was as efficient as transfer of other EHEC chromosomalmarkers. One Gal+ revertant strain (TEA040) was chosen to test for P1sensitivity. Like the isogenic wild type strain, this Gal+ revertant hada reduced capacity to adsorb P1 phage compared to the gal mutants (Table2), was resistant to P1 lysis in a P1 cross-streak experiment, and wasnot able to serve as a recipient for P1 transduction. Nonetheless,knowledge of the mechanism is not required in order to make and use thepresent invention.

An O157:H7 galETKM mutant is dramatically impaired in colonization ofthe infant rabbit intestine. Previous studies have shown that galEmutants of S. enterica serovars are defective in intestinal colonizationassays (Gilman et al., J Infect Dis, 136:717-723, 1977; and Hohmann etal., Infect Immun, 25:27-33, 1979). To determine whether the EHECgalETKM deletion had a similar defect in intestinal colonization, thegalETKM:aad-7 deletion mutant (TEA026) was tested in a competition assayagainst the isogenic wild type strain (EDL933) using the EHEC-infantrabbit model. The galETKM deletion mutant (TEA026) was ˜500-fold lessable to colonize the infant rabbit ileum, cecum, and mid-colon (FIG.1A). To demonstrate that this dramatic defect in intestinal colonizationis due to the galETKM deletion, the Gal+ reverted strain was placed incompetition against its ΔgalETKM::aad-7 parent strain. The Gal+revertant (TEA040) out-competed the galETKM mutant (TEA023) to a similarextent as the wild type (FIG. 1B), demonstrating that the galETKMdeletion accounts for the colonization defect of TEA026. In vitrocompetition assays where the galETKM strain and the wild type or theGal+ revertant were grown in LB at 37° C. revealed that the galETKMmutation conferred a slight (˜2-fold) but statistically significantgrowth defect in rich medium (FIG. 1). This minor in vitro growth defectcannot account for the drastic colonization defect of the gal mutant.Overall these findings indicate that the O157 O-antigen is critical forEHEC intestinal colonization. Nonetheless, knowledge of the mechanism isnot required in order to make and use the present invention.

O157:H7 gal mutants are more sensitive to BPI killing. Host organismsoften produce anti-microbial peptides, such as cryptidins, as effectorsof innate immunity. Bactericidal/permeability-increasing protein (BPI)is a 55- to 60-kDa protein that is found in the blood and on surfaces ofepithelial cells throughout the gastrointestinal tract (Canny et al.,Proc Natl Acad Sci USA, 99:3902-3907, 2002). BPI binds the lipid Acomponent of LPS and has potent bactericidal activity againstgram-negative bacteria (Gazzano-Santoro et al., Infect Immun,60:4754-4761, 1992). Given that O157 gal mutants likely lack O-antigen,it was contemplated that these mutants would be more sensitive to BPIthan the wild type strain, since the lipid A portion of the gal strains'LPS would be more accessible to BPI binding. P2, a peptide that containsBPI residues 86 to 104 and which has the same cytotoxic activity as theentire protein was used to test whether the O157:H7 gal mutants haveincreased susceptibility to BPI. Bacteria were incubated in the presenceor absence of P2 before enumerating the bacteria. Each of the galmutants was far more sensitive to P2 than the wild type EHEC strain orthe Gal+ revertant (Table 2). These data indicate that O157 O-antigen isimportant for EHEC resistance to BPI. Nonetheless, knowledge of themechanism is not required in order to make and use the presentinvention.

Example 2 Production of E. coli O157:H7 gal stx2 Mutants

This example describes the construction of O-antigen deficient, shigatoxin 2 deficient E. coli O157:H7 mutants. The E. coli O157:H7 gal stx2mutants are contemplated to poorly colonize the intestine in vivo, causeonly mild diarrhea and result in reduced intestinal inflammation incomparison to isogenic E. coli O157:H7.

Bacterial Strain Construction. E. coli O157:H7 gal stx2 mutants areconstructed using the PCR based “one-step gene inactivation system”adapted from Datsenko and Wanner (Proc Nail Acad Sci USA, 97:6640-6645,2000). The pKD4 plasmid is used as a template to amplify a kanamycinresistance gene for these studies. PCR primers are designed using DNAsequences derived from E. coli O157:H7 reference strain EDL933 per apublished report (Ritchie et al., Infect Immun, 71:7129-7139, 2003) andas follows: for stx_(2AB), JRW1 (5′-ATGAAGTGTA TATTATTTAA ATGGGTACTGTGCCTGGTGT AGGCTGGAGC TGCTTCG-3′ set forth as SEQ ID NO:10) and JRW2(5′-TTATGCCTCA GTCATTATTA AACTGCACTT CAGCAACATA TGAATATCCT CCTTA-3′ setforth as SEQ ID NO:11). PCR products were electroporated into TEA026(ΔgalETKM::aad-7) and TEA028 (ΔgalETKM::tetA) previously transformedwith the lambda Red-encoding plasmid pKD46, to makeΔgalETKM::aad-7/Δstx2ab::kan and ΔgalETKM::tetA/Δstx2ab::kan,respectively. Recombinants containing the kanamycin resistance gene inplace of the gene of interest are selected on L-agar plates containing50 μg/ml kanamycin, and the deletion of the gene of interest wasconfirmed by PCR analyses. The growth rates of new strains are notcontemplated to differ from that of EDL933 during in vitro growth in LBat 37° C.

Animal protocols. Litters of 2-day-old New Zealand White rabbits areobtained from a commercial breeding company (Pine Acre Rabbitry, Norton,Mass.). Each litter is housed as a group and nursed by the mother.Three-day-old rabbits are intragastrically inoculated with E. colimutant strains of interest or phosphate-buffered saline (PBS) using size5 French catheters with flexible tips (Arrow International, Reading,Pa.). Bacterial doses of 5×10⁸ CFU per 90 g of rabbit body weight areused in most experiments. For infant rabbit experiments, bacteria aregrown overnight in LB at 37° C., harvested by centrifugation, and thenresuspended in sterile PBS (pH 7.2) and adjusted to a cell density ofabout 10⁹ CFU/ml. Postinoculation, the infant rabbits are weighed dailyand observed twice daily for clinical signs of illness. Diarrhea isscored as follows: none, no diarrhea (normal pellets are dark green,hard, and formed); mild, diarrhea consisting of a mix of softyellow-green unformed and formed pellets, resulting in light staining ofthe hind legs; severe, diarrhea consisting of unformed or liquid stool,resulting in significant staining of the perineum and hind legs. In mostexperiments, rabbits are necropsied 7 days postinoculation. All rabbitswere necropsied by intracardiac injection with 1 ml of saturated KClsolution following isoflurane anesthesia (Aerrane, Baxter, Deerfield,Ill.). At necropsy, the intestinal tract from the duodenum to the anusis removed and samples are obtained for histologic and microbiologicanalyses. To limit any litter-specific effects, at least two differentlitters are used to test each type of inoculum studied.

Histology. Tissues are fixed in 10% neutral-buffered formalin, routinelyprocessed for histology, and stained with hematoxylin and eosin (H&E).The samples are semi-quantitatively assessed for infiltration ofheterophils, mononuclear cells, and edema or congestion by a comparativepathologist blinded to the sample identity. Sections are evaluated forheterophil inflammation using the following criteria: 0, none; 1,scattered individual cells or small clusters limited to the superficiallamina propria; 2, multifocal aggregates involving the entire mucosasurface with small numbers in the lumen; 3, coalescing heterophilicmucosal inflammation with abundant cell extrusion into the lumen; and 4,necrotizing inflammation with ulceration, large heterophilicintraluminal rafts, and extension into submucosal and deeper layers.Sections are evaluated for mononuclear cells (predominantly lymphocytesand plasmacytes) using the following criteria 0, normal; 1, slightlyincreased numbers in the lamina propria; 2, moderately increased numberswith mild separation of the crypts; 3, markedly increased mononuclearcells with decreased crypts and prominent intramucosal follicles; 4,effacing mononuclear cell inflammation with large mucosal and/orsubmucosal follicles ±extension into deeper layers. Edema, congestion,and hemorrhage are subjectively evaluated as follows: 0, none; 1, mildvascular congestion and/or edema limited to the lamina propria; 2,moderate, involving both mucosa and submucosa; 3, severe congestion andedema f small hemorrhages of mucosa and submucosa and edema of theserosa; and 4, severe diffuse transmural congestion, edema, andmultifocal hemorrhage. Samples for transmission electron microscopy fromthe ceca and distal colons of rabbits necropsied 2 days postinoculationare fixed in 2.5% glutaraldehyde (pH 7.3) buffered in 0.1 M sodiumcacodylate. Ultrathin sections of these samples are then stained withuranyl acetate and lead citrate, post fixed with osmium tetroxide,before examination on a Phillips CM-10 transmission electron microscope.

Example 3 Immunization with E. coli O157:H7 gal stx2 Mutants

This example describes the administration of O-antigen deficient, shigatoxin 2 deficient E. coli O157:H7 mutants to a subject under conditionssuitable for induction of an E. coli O157:H7-reactive immune response.The methods described herein are adapted from US Publication No.2002/0160020 of Finlay and Potter, herein incorporated by reference.

Experimental Animals. Cattle, between the ages of 8 and 12 months arepurchased from local ranchers. Fecal samples are obtained daily fromeach animal for 14 days. The number of EHEC O157:H7 in the fecal samplesis determined by plating on Rainbow Agar. The plates are incubated at37° C. for 2 days and black colonies are enumerated. Growth is scoredfrom 0-5. Animals having a score of 0 (no EHEC O157:H7) are used in allexperiments.

Immunization. Sixteen cattle are divided in two groups of eight animalswith group 1 receiving a composition comprising live E. coli O157:H7 galstx2 mutants and group 2 receiving excipient alone by oral-gastricintubation on day O, Seroconversion is assayed using an enzyme-linkedimmunosorbent assay (ELISA) on days 0 (pre-immunization), 14, 21 and 28.Briefly, EHEC O157:H7 is used to coat the wells of microtiter platesovernight at 4° C. The wells are washed, and then blocked with 0.5%nonfat dried milk in PBS. Serial dilutions of sera are added to eachwell and incubated for 2 hr at 37° C. The wells are washed beforeincubation with a 1:5000 dilution of peroxidate-conjugated rabbitanti-bovine immunoglobulin M, G and A for 1 hr at 37° C. Cattle of group1 are contemplated to develop high-titer anti-EHEC antibody responses.

Example 4 Challenge of E. coli O157:H7 gal stx2 Mutant-ImmunizedSubjects

This example describes the effects of immunizing a mammalian subjectwith O-antigen deficient shiga toxin 2 deficient E. coli O157:H7 mutantson intestinal colonization and fecal shedding of an EHEC challengestrain deficient in shiga toxin 2.

EHEC Challenge. At day 36, Group 1 and Group 2 animals of Example 3 arechallenged with approximately 10⁸ CFU of EHEC O157:H7 by oral-gastricintubation. Fecal shedding is monitored for 14 days. Briefly, fecalmaterial is weighed, suspended in sterile saline and inoculated intoculture media. After overnight growth, bacteria are replica plated on LBagar containing spectinomycin (100 μg/ml) to determine the number ofchallenge bacteria. Fewer experimental animals are contemplated to shedEHEC O157:H7 than control animals. Moreover, experimental animals thatshed EHEC O157:H7 are contemplated to do so for a shorter period of timethan control animals. In addition the total number of EHEC O157:H7bacteria isolated from fecal samples is contemplated to be significantlylower among the EHEC-vaccinated group as compared to the placebo group.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention, which are obvious to those skilled inmolecular biology, genetics, microbiology, immunology or related fieldsare intended to be within the scope of the following claims.

1. An isolated Escherichia coli (E. coli) bacterium comprising aninactivating mutation in one or more galactose-modifying enzymes.
 2. TheE. coli bacterium of claim 1, wherein said galactose-modifying enzymesare selected from the group consisting of GalE, GalT, GalK and GalU. 3.The E. coli bacterium of claim 1, wherein said E. coli is selected fromthe group consisting of enterohemorrhagic E. coli (EHEC),enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) anduropathogenic E. coli (UPEC).
 4. The E. coli bacterium of claim 1,wherein said E. coli is enterohemorrhagic E. coli (EHEC).
 5. The E. colibacterium of claim 4, wherein said EHEC is E. coli O157.
 6. The E. colibacterium of claim 5, wherein said E. coli O157 is serotype O157:H7. 7.The E. coli bacterium of claim 5, wherein said E. coli O157 is of astrain selected from the group consisting of TEA007 (galE::pTHE001),TEA023 (ΔgalU::aad-7), TEA026 (ΔgalETKM::aad-7) and TEA028(ΔgalETKM::tetA).
 8. The E. coli bacterium of claim 1, wherein saidinactivating mutation is associated with reduced O-antigen expression.9. The E. coli bacterium of claim 1, wherein said inactivating mutationis associated with increased susceptibility to bacteriophage P1.
 10. TheE. coli bacterium of claim 1, wherein said inactivating mutation isassociated with increased sensitivity tobactericidal/permeability-increasing protein (BPI).
 11. The E. colibacterium of claim 10, wherein said BPI comprises the amino acidsequence of peptide P2 set forth as SEQ ID NO:9.
 12. The E. colibacterium of claim 1, wherein said inactivating mutation is associatedwith reduced intestinal colonization.
 13. The E. coli bacterium of claim1 further comprising an inactivating mutation in a shiga toxin Asubunit.
 14. The E. coli bacterium of claim 13, wherein said shigatoxinA subunit is selected from the group consisting of Stx1A and Stx2A. 15.A composition comprising the E. coli bacterium of claim 14 and anadjuvant or an excipient.
 16. A method for inducing an immune response,comprising administering the composition of claim 15 to a subject underconditions suitable for inducing an immune response against said E. colibacterium.
 17. The method of claim 16, wherein said administering isdone orally or intrarectally.
 18. The method of claim 16, wherein saidsubject is a human.
 19. The method of claim 16, wherein said subject isa ruminant.
 20. The method of claim 19, wherein said ruminant is abovine subject.